The identification of biomarkers to track clinical activity of drugs and as predictive tools for patient selection is critical in our quest to develop personalized cancer therapies. This is especially critical in the field of cancer immunotherapy, where complex interacting factors ultimately control treatment efficacy and where responses can occur over a protracted period of time. Adoptive cell therapy using expanded autologous tumor-infiltrating lymphocytes (TIL) together with IL-2 has emerged as a powerful salvage therapy for metastatic melanoma. Multiple TIL therapy clinical trials, including those at our center, have consistently yielded objective tumor regression rates and prolonged survival in about 50% of patients that have progressed after multiple previous therapies, including targeted therapies (BRAF and MEK inhibition) and newer immunotherapies, such as CTLA-4 and PD-1 blockade. However, 3 major gaps in our knowledge limit our ability to further develop TIL therapy as a mainstream therapeutic: 1) We still know relatively little about the types of T cells n TIL mediating tumor regression and how their levels change in vivo paralleling changes in tumor regression and relapse, 2) There have been no in-depth biomarker studies on host tumor and blood factors in TIL therapy and how they are related to clinical response, and 3) Nothing is known about the mechanisms of resistance within tumors that do not respond to TIL therapy, especially during secondary progression (relapse) after an initial response. In this project, we propose to perform a comprehensive biomarker study on melanoma patients getting TIL therapy. We hypothesize that a number of interacting immunoregulatory factors within the tumor microenvironment, together with systemic inflammatory mediators and tumor progression factors regulate TIL phenotype and can distinguish patients responding to TIL therapy and those who develop resistance (tumor escape) during therapy. Here for the first time, we will combine a number of different synergistic approaches to identify biomarkers in the expanded TIL, tumor, and blood that are predictive of clinical response. In Aim #1, we will analyze melanoma TIL in tumors and after ex vivo expansion and their association with clinical response and TIL persistence in vivo. In Aim #2, we will measure markers of inflammation, immune suppression, and tumor progression in tumor tissue and blood as predictive markers of response to TIL therapy. In Aim #3, by acquiring additional tumor and blood samples from patients following their initial TIL infusion, we will explore the mechanisms of resistance in tumors that initially d not respond, or in new sites of metastases that are surgically removed. These studies will not only identify novel factors that can be targeted to further improve TIL therapy, but have implications for all forms of immunotherapy that ultimately converge on the function of TIL in the tumor microenvironment and the interplay of factors that facilitate or suppress T-cell infiltration and function at the tumor site.